Ciliates are unicellular yet possess much of the cellular complexity found in multicellular organisms. They have evolved substantial biological innovations (e.g. nuclear dimorphism, programmed DNA rearrangements), the studies of which have revealed fundamental insights into living systems. Even so, they remain an understudied class of microbes. Tetrahymena in particular has had huge impact on biology, including the discovery of ribozymes and telomerase, and continues to contribute to our understanding of basic mechanisms critical to human health and disease. The Tetrahymena Stock Center is the primary resource for the maintenance and distribution of genetically defined strains of Tetrahymena thermophila to researchers in this country and abroad. This project aims to develop core new technologies to manipulate the genome of Tetrahymena and thereby enable researchers to more adeptly contribute to the basic understanding of cellular processes. A major goal of the project is to adapt CRISPR-Cas9-mediated genome editing for use in Tetrahymena, specifically to promote more efficient modification of the germline genome. Targeting of Cas9 and guide RNAs specifically to the micronucleus will lead to specific alterations of this silent germline nucleus rather than that of the expressed somatic nucleus. Specific modification of the silent germline will allow Tetrahymena researchers to create and subsequently study deleterious mutations when they are brought into the expressed nucleus in the next generation. A major goal for this applied research is to lower barriers that impede the use of Tetrahymena to study fundamental biology and model human disease. Tetrahymena also offers many advantages as a protein expression system. To improve the stability of high level expression from ribosomal DNA-based expression vectors, the project aims create cell lines deleted for the single-copy rRNA gene in the germline and thereby eliminate competition between the endogenous rDNA and introduced expression vectors, which will provide for more reliable protein production. Development of these approaches will enable mutagenesis and vector-based transformation without the need for drug selection. Successful attainment of these goals will assist numerous scientist is their investigations. These methodologies and associated resources will be distributed through the Stock Center and will be used to expand the services offered by the Center to the research community with an additional goal of increasing the Center?s sustainability.